2A: We asked the question: what are the changes in tyrosine phosphorylation of proteins upon EGF stimulation and tyrosine kinase inhibitor (TKI) inhibition in human lung adenocarcinoma cell lines expressing the mutant EGFRs? We used two TKIs, erlotinib, a reversible EGFR inhibitor and BIBW2992, an irreversible EGFR and ERBB2 inhibitor. Various large-scale experiments were performed using SILAC and mass spectrometry. Lung adenocarcinoma cell lines used in these experiments were H3255 and 11-18 (L858R mutation), H1975 (L858R/T790M mutation), PC9 (E746-A750 Del EGFR). In addition isogenic NR6 (a variant of 3T3 fibroblasts) and HBECs (human bronchial epithelial cells) with stable expression of WT EGFR, L858R EGFR, Del EGFR were also used for phosphorylation studies. 200-700 proteins have been identified from each of the above experiments and their relative quantitation performed. Currently we are validating the change in phosphorylation of a subset of these proteins by immunoprecipitation and western blot experiments. We also performed siRNA-mediated knockdown of proteins identified as phosphorylation targets of mutant EGFRs in lung adenocarcinoma cells harboring mutant EGFRs or mutant KRAS. It is interesting to note that several of the proteins whose tyrosine phosphorylation was inhibited by TKIs in mutant EGFR-expressing cells were also required for survival of EGFR mutant expressing cells but not KRAS mutant expressing cells. Examples of such targets that we are following more are EPHA2, SCAMP3, DAPPI1. Some of these proteins such as DAPPI1 (dual adapter for phosphotyrosine or 3-phosphoinositides) has not been implicated in EGFR signaling. However tyrosine phosphorylation at Y139 of DAPPI1 is stimulated upon EGF stimulation and inhibited 5-10 fold upon treatment with TKIs, erlotinib and BIBW2992, suggesting DAPPI1 may be an integral member of the EGFR signaling pathway. 2B: Identification of Ser/Thr phosphorylation sites on downstream targets of mutant EGFRs and quantitation of phosphorylation changes upon TKI inhibition. We initiated experiments to identify Ser/Thr phosphorylation sites by SILAC labeling of adenocarcinoma cells and mass spectrometry. We used various fractionation techniques after in-solution tryptic digestion such as strong cation exchange (SCX) or basic reverse phase. Around 30 fractions were then subjected to TiO2 enrichment for phosphopeptide isolation followed by reverse phase liquid chromatography and tandem mass spectrometry using an orbitrap velos mass spectrometer. We continued our previous collaboration with Dr. Akhilesh Pandey at Johns Hopkins to perform the mass spectrometry analysis. H3255 lung adenocarcinoma cells harboring the L858R mutation were used in a triple-SILAC experiment. A total of 3,586 phosphosites (3167 phosphoserine, 395 phosphothreonine and 24 phosphotyrosine sites) were identified from this study, which corresponds to 1,434 proteins. There were 278 phosphosites that were activated upon EGF treatment and 358 that were dephosphorylated. Majority of phosphosites (2673 sites) did not show any change on treatment with EGF. This observation is reflective of the existence of very specific signaling cascades that are disrupted on treatment with the drug. We are now analyzing this data set using several bioinformatic tools, including IPA and Ariadne pathway studio. Various canonical pathways such as p70S6, IRS, ERK/MAPK, mTOR, PKA, JAK/STAT were enriched in our dataset.